title: Development of genetically modified eliminable human dermal fibroblast feeder cells for ocular surface regeneration medicine.
abstract: OBJECTIVE
  Cultured human corneal limbal stem/progenitor cells are usually established and maintained on feeder layers. However, animal feeder cells are associated with viral infection, pathogen transmission, and xenogenic contamination. All feeder cells also can be mixed easily into cell-sheet production, causing self-contamination. We developed a line of labeled, immortalized, eliminable human dermal fibroblast cells to eliminate these problems.
  
  
  METHODS
  The enhanced green fluorescent protein gene, human-derived telomerase reverse transcriptase gene, and herpes simplex virus thymidine kinase gene were transfected into human dermal fibroblast cells to establish labeled, immortalized, eliminable feeder cells. Established eliminable dermal fibroblasts (TERT+TK-D) were treated with mitomycin, cocultured with human limbal stem/progenitor cells to regenerate epithelium sheets, and compared with 3T3 feeder cells.
  
  
  RESULTS
  Established TERT+TK-D feeder cells maintained immortalization, visualization, and eliminable characteristics during 6 months of continuous passages. The colony-forming efficiency of limbal stem/progenitor cells was similar in the TERT+TK-D group (11.77 ± 0.21%) and the 3T3 group (12.8 ± 1.61%) (P = 0.332). All cell sheets were well stratified into 4 to 5 layers. The TERT+TK-D group colonies and epithelial cell sheets showed weaker staining of corneal epithelium differentiation marker K3 than the 3T3 group and quantitative analysis of mRNA transcripts. Moreover, PCR analysis against the long terminal repeat sequence of the lentiviral vector integrated into the genetically modified feeder cells showed no contamination of ganciclovir-treated regeneration epithelial sheets.
  
  
  CONCLUSIONS
  Genetically modified, labeled, immortalized, eliminable human dermal feeder cells are promising substitutes for 3T3 feeder cells for xenogeny-free ocular surface regeneration.
authors.affiliation: Department of Ophthalmology, Osaka University Medical School, Suita, Japan.
authors.forename: Yingli
authors.initials: Y
authors.lastname: Li
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Inoue
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Takamatsu
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Maeda
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Ohashi
authors.forename: Kohji
authors.initials: K
authors.lastname: Nishida
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015415:Biomarkers; D002460:Cell Line; D018920:Coculture Techniques; D060527:Corneal Keratocytes / Q000166:cytology / Q000378:metabolism; D020405:Dermis / Q000166:cytology*; D019573:Epithelium, Corneal / Q000502:physiology*; D061252:Feeder Cells; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015774:Ganciclovir / Q000633:toxicity; D015870:Gene Expression / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D051379:Mice; D012038:Regeneration / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018139:Simplexvirus / Q000201:enzymology; D013234:Stem Cells / Q000166:cytology*; D019098:Telomerase / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection
chemical_list: D015415:Biomarkers; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D019098:Telomerase; D015774:Ganciclovir
keywords: corneal epithelium; eliminable feeder cell; human dermal fibroblast